Fitc anti annexin 5

For ex vivo stimulation, iNKT cells need to be enriched from thymocytes and splenocytes by depleting CD8α⁺ cells. Briefly, the total thymocytes in 200 μL of Hank's Balanced Salt (HBSS) were incubated with CD8α (Ly-2) MicroBeads (Miltenyi Biltec) on ice for 15 min, and iNKT cells were enriched using LS columns (Miltenyi Biltec) according to the manufacturer's protocol. The enriched cells were used for surface staining of PE-anti-ICOS (TE.17G9, eBioscience), PE-anti-IL-23R (12B2B64), and FITC-anti-Annexin V (Biolegend). Intracellular staining for PLZF, RORγt, T-bet, GATA3, Bcl2, Ki-67, and c-Maf was fixed and permeabilized using a Foxp3 Staining Buffer Set (eBiosciense). For α-GalCer stimulation, thymocytes (6 × 10⁶) and splenocytes (6 × 10⁶) were seeded in a 24-well plate in 1640+10% FBS, left unstimulated, or stimulated with α-GalCer (125 ng/ml) for 72 h, with the addition of PMA (50 ng/ml) and ionomycin (500 ng/ml)in the last 5 h. After stimulation, cells were stained with CD1d, TCRβ, CD44, NK1.1, IFN-γ, IL-17a, IL-4, and TNFα.

For flow cytometry, we used FITC anti-CD4, APC anti-CD8, FITC anti-IgM, APC anti-CXCR5, PE anti-PD1, FITC anti-CD62L, PE anti-CD25, APC anti-LAG-3, APC anti-TIM-3, PE anti-PD-1 and FITC anti-ICOS, FITC anti-Annexin V and APC anti-FoxP3 antibodies (all from BioLegend).